Primer Extension for Mitochondrial RNA Analysis

Primer extension was conducted basically as described (27 (link),28 (link)). The 5΄ ³²P-labeled primer (0.1 pmol) was incubated with 3.125 μg total RNA in a 5 μl mixture containing 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 2 min at 80°C, followed by cooling to room temperature. Subsequently, 0.5 μl (100 units) SuperScript III (Invitrogen) was added to 4.5 μl of the premixed solution [2 μl 5 × FS buffer (Invitrogen), 0.25 μl of 1.5 mM d/ddNTP mix, 0.75 μl ddH₂O and 1.5 μl of 25 mM MgCl₂]. The d/ddNTP mix consisted of dTTP, dGTP and ddCTP for mt-tRNA^Lys; dTTP, dGTP and ddATP for mt-tRNA^Arg; and dATP, dTTP, dCTP and ddGTP for mitochondrial 16S rRNA. The reaction was carried out for 1 h at 47°C for mt-tRNAs and 55°C for mitochondrial 16S rRNA. The reaction was stopped by addition of 0.5 μl of 4 M NaOH and boiling for 5 min at 95°C, and then neutralized by addition of 4.5 μl of 1 M Tris–HCl (pH 5.0). The cDNAs were resolved by 20% denaturing PAGE. The gel was exposed to an imaging plate (BAS-MS2040, Fujifilm) and visualized on a FLA-7000 fluorimager (Fujifilm).